微小RNA-1a-3p在小鼠神经源性膀胱中的作用及其机制

Action mechanism of microRNA-1a-3p in mouse neurogenic bladder

目的建立小鼠神经源性膀胱动物模型,探讨纤维连接蛋白1(FN1)的变化及微小RNA(miRNA,miR)-1a-3p的作用机制。方法C57BL/6J雌性7周龄小鼠40只(购自北京维通利华实验动物技术有限公司),分为模型组和对照组,模型组皮下注射髓鞘少突胶质细胞糖蛋白及结核分支杆菌,并48 h腹腔注射百日咳毒素;对照组皮下注射生理盐水。观察小鼠排尿频次、排尿量等变化。处死小鼠并取膀胱组织,模型组和对照组各选取3只膀胱进行高通量测序。采用实时定量反转录聚合酶链反应(RT-qPCR)、蛋白印迹法(Western blot)检测小鼠膀胱组织中FN1和miR-1a-3p的变化。多组计量资料采用One-way ANOVA。结果对测序结果进行分析,随着泌尿系统症状加重,FN1增加(4.569±0.426,t=-5.142,P<0.01),而miR-1a-3p减少(1.623±0.312,t=-3.945,P<0.05),差异有统计学意义。与对照组比较,模型组膀胱组织中FN1的mRNA[2.501(1.301,4.251),F=99.183,P<0.01]和蛋白[2.937(1.174,4.262),F=63.834,P<0.01]表达均上调,差异有统计学意义,而miR-1a-3p[2.401(1.250,3.001),F=35.951,P<0.01]表达下调,差异有统计学意义;双荧光素酶基因报告表明FN1为miR-1a-3p的直接靶基因(0.514±0.027,t=13.355,P<0.01),差异有统计学意义。结论神经源性膀胱动物模型出现尿频、尿急、尿潴留等症状,且FN1表达明显上调,而miR-1a-3p表达下调,因此miR-1a-3p可能通过对FN1调控参与神经源性膀胱发生发展。

Objective To establish a mouse model of neurogenic bladder and to investigate the changes of fibronectin 1(FN1)and the action mechanism of microRNA(miRNA,miR)-1a-3p.Methods Mice were divided into model group and control group.The model group received subcutaneous injection of myelin oligodendrocyte glycoprotein and mycobacterium tuberculosis,and intraperitoneal injection of pertussis toxin at 48 h.The control group received subcutaneous injection of normal saline.The frequency of urination and the amount of urine output were observed.The mice were sacrificed and the bladder tissue was taken.Three bladders were selected from the model group and the control group for high-throughput sequencing.The changes of FN1 and miR-1a-3p in mouse bladder tissues were detected by real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR)and Western blotting.Results The sequencing results were analyzed.As the urinary system symptoms worsened,FN1 increased(4.569±0.426,t=-5.142,P<0.01),while miR-1a-3p decreased(1.623±0.312,t=-3.945,P<0.05).Compared with the control group,the expression of FN1 mRNA[2.501(1.301,4.251),F=99.183,P<0.01]and protein[2.937(1.174,4.262),F=63.834,P<0.01]in the bladder tissue of the model group was up-regulated,and that of miR-1a-3p was down-regulated[2.401(1.250,3.001),F=35.951,P<0.01].The dual luciferase gene report indicated that FN1 was a direct target gene of miR-1a-3p(0.514±0.027,t=13.355,P<0.01).Conclusion The animal model of neurogenic bladder has urinary frequency,urgency,urinary retention and other symptoms,and the expression of FN1 is up-regulated,while the expression of miR-1a-3p is down-regulated.Therefore,miR-1a-3p may participate in neurogenic bladder formation through regulation of FN1.